scholarly journals Physiological Conditions and dsRNA Application Approaches for Exogenously induced RNA Interference in Arabidopsis thaliana

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 264
Author(s):  
Konstantin V. Kiselev ◽  
Andrey R. Suprun ◽  
Olga A. Aleynova ◽  
Zlata V. Ogneva ◽  
Alexandra S. Dubrovina
Keyword(s):  
Arabidopsis Thaliana ◽  
Soil Moisture ◽  
Rna Interference ◽  
Plant Pathogens ◽  
Foliar Application ◽  
Time Of Day ◽  
Plant Age ◽  
Late Time ◽  
Small Interfering Rnas ◽  
Physiological Conditions

Recent studies have revealed that foliar application of double-stranded RNAs (dsRNAs) or small-interfering RNAs (siRNAs) encoding specific genes of plant pathogens triggered RNA interference (RNAi)-mediated silencing of the gene targets. However, a limited number of reports documented silencing of plant endogenes or transgenes after direct foliar RNA application. This study analyzed the importance of physiological conditions (plant age, time of day, soil moisture, high salinity, heat, and cold stresses) and different dsRNA application means (brush spreading, spraying, infiltration, inoculation, needle injection, and pipetting) for suppression of neomycin phosphotransferase II (NPTII) transgene in Arabidopsis thaliana, as transgenes are more prone to silencing. We observed a higher NPTII suppression when dsRNA was applied at late day period, being most efficient at night, which revealed a diurnal variation in dsRNA treatment efficacy. Exogenous NPTII-dsRNA considerably reduced NPTII expression in 4-week-old plants and only limited it in 2- and 6-week-old plants. In addition, a more discernible NPTII downregulation was detected under low soil moisture conditions. Treatment of adaxial and abaxial leaf surfaces by brushes, spraying, and pipetting showed a higher NPTII suppression, while infiltration and inoculation were less efficient. Thus, appropriate plant age, late time of day, low soil moisture, and optimal dsRNA application modes are important for exogenously induced gene silencing.

scholarly journals External dsRNA Downregulates Anthocyanin Biosynthesis-Related Genes and Affects Anthocyanin Accumulation in Arabidopsis thaliana

2021 ◽  
Vol 22 (13) ◽  
pp. 6749
Author(s):  
Konstantin V. Kiselev ◽  
Andrey R. Suprun ◽  
Olga A. Aleynova ◽  
Zlata V. Ogneva ◽  
Alexander V. Kalachev ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Plant Pathogens ◽  
Foliar Application ◽  
Anthocyanin Biosynthesis ◽  
Plant Biotechnology ◽  
Laser Scanning Microscopy ◽  
Mrna Levels ◽  
Anthocyanin Accumulation ◽  
Small Interfering Rnas ◽  
Functional Studies

Exogenous application of double-stranded RNAs (dsRNAs) and small-interfering RNAs (siRNAs) to plant surfaces has emerged as a promising method for regulation of essential genes in plant pathogens and for plant disease protection. Yet, regulation of plant endogenous genes via external RNA treatments has not been sufficiently investigated. In this study, we targeted the genes of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, and two transcriptional factors, MYBL2 and ANAC032, negatively regulating anthocyanin biosynthesis in Arabidopsis. Direct foliar application of AtCHS-specific dsRNAs and siRNAs resulted in an efficient downregulation of the AtCHS gene and suppressed anthocyanin accumulation in A. thaliana under anthocyanin biosynthesis-modulating conditions. Targeting the AtMYBL2 and AtANAC032 genes by foliar dsRNA treatments markedly reduced their mRNA levels and led to a pronounced upregulation of the AtCHS gene. The content of anthocyanins was increased after treatment with AtMYBL2-dsRNA. Laser scanning microscopy showed a passage of Cy3-labeled AtCHS-dsRNA into the A. thaliana leaf vessels, leaf parenchyma cells, and stomata, indicating the dsRNA uptake and spreading into leaf tissues and plant individual cells. Together, these data show that exogenous dsRNAs were capable of downregulating Arabidopsis genes and induced relevant biochemical changes, which may have applications in plant biotechnology and gene functional studies.

scholarly journals Influence of Land Cover and Soil Moisture on the Horizontal Distribution of Sensible and Latent Heat Fluxes in Southeast Kansas during IHOP_2002 and CASES-97

2007 ◽  
Vol 8 (1) ◽  
pp. 68-87 ◽  
Author(s):  
Margaret A. LeMone ◽  
Fei Chen ◽  
Joseph G. Alfieri ◽  
Mukul Tewari ◽  
Bart Geerts ◽  
...  
Keyword(s):  
Soil Moisture ◽  
Latent Heat ◽  
Land Surface ◽  
Field Experiments ◽  
Time Of Day ◽  
Horizontal Distribution ◽  
Heat Fluxes ◽  
Surface Model ◽  
Green Vegetation ◽  
The Impact

Abstract Analyses of daytime fair-weather aircraft and surface-flux tower data from the May–June 2002 International H2O Project (IHOP_2002) and the April–May 1997 Cooperative Atmosphere Surface Exchange Study (CASES-97) are used to document the role of vegetation, soil moisture, and terrain in determining the horizontal variability of latent heat LE and sensible heat H along a 46-km flight track in southeast Kansas. Combining the two field experiments clearly reveals the strong influence of vegetation cover, with H maxima over sparse/dormant vegetation, and H minima over green vegetation; and, to a lesser extent, LE maxima over green vegetation, and LE minima over sparse/dormant vegetation. If the small number of cases is producing the correct trend, other effects of vegetation and the impact of soil moisture emerge through examining the slope ΔxyLE/ΔxyH for the best-fit straight line for plots of time-averaged LE as a function of time-averaged H over the area. Based on the surface energy balance, H + LE = Rnet − Gsfc, where Rnet is the net radiation and Gsfc is the flux into the soil; Rnet − Gsfc ∼ constant over the area implies an approximately −1 slope. Right after rainfall, H and LE vary too little horizontally to define a slope. After sufficient drying to produce enough horizontal variation to define a slope, a steep (∼−2) slope emerges. The slope becomes shallower and better defined with time as H and LE horizontal variability increases. Similarly, the slope becomes more negative with moister soils. In addition, the slope can change with time of day due to phase differences in H and LE. These trends are based on land surface model (LSM) runs and observations collected under nearly clear skies; the vegetation is unstressed for the days examined. LSM runs suggest terrain may also play a role, but observational support is weak.

scholarly journals Analysis of the Interaction of Primate Retroviruses with the Human RNA Interference Machinery

2007 ◽  
Vol 81 (22) ◽  
pp. 12218-12226 ◽  
Author(s):  
Jennifer Lin ◽  
Bryan R. Cullen
Keyword(s):  
Rna Interference ◽  
Virus Type ◽  
Leukemia Virus ◽  
Foamy Virus ◽  
Human Cells ◽  
Small Interfering Rnas ◽  
Cell Leukemia Virus Type ◽  
Human T Cell ◽  
Hiv 1

ABSTRACT The question of whether retroviruses, including human immunodeficiency virus type 1 (HIV-1), interact with the cellular RNA interference machinery has been controversial. Here, we present data showing that neither HIV-1 nor human T-cell leukemia virus type 1 (HTLV-1) expresses significant levels of either small interfering RNAs or microRNAs in persistently infected T cells. We also demonstrate that the retroviral nuclear transcription factors HIV-1 Tat and HTLV-1 Tax, as well as the Tas transactivator encoded by primate foamy virus, fail to inhibit RNA interference in human cells. Moreover, the stable expression of physiological levels of HIV-1 Tat did not globally inhibit microRNA production or expression in infected human cells. Together, these data argue that HIV-1 and HTLV-1 neither induce the production of viral small interfering RNAs or microRNAs nor repress the cellular RNA interference machinery in infected cells.

scholarly journals Human Immunodeficiency Virus Type 1 Escapes from RNA Interference-Mediated Inhibition

2004 ◽  
Vol 78 (5) ◽  
pp. 2601-2605 ◽  
Author(s):  
Atze T. Das ◽  
Thijn R. Brummelkamp ◽  
Ellen M. Westerhout ◽  
Monique Vink ◽  
Mandy Madiredjo ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Interference ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Antiviral Drug ◽  
Target Sequence ◽  
Small Interfering Rnas ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses.

scholarly journals Small interfering RNAs are highly effective inhibitors regarding Crimean-Congo hemorrhagic fever virus replication in vitro

2020 ◽  
Author(s):  
Fanni Földes ◽  
Mónika Madai ◽  
Henrietta Papp ◽  
Gábor Kemenesi ◽  
Brigitta Zana ◽  
...  
Keyword(s):  
Rna Interference ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
World Health ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Antiviral Therapies

AbstractCrimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of World Health Organization, considering its potential to create a public health emergency and more importantly, the absence of efficacious drugs and/or vaccines regarding treatment. The highly lethal nature characteristic to CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design siRNAs that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 hours, nucleic acid from the supernatant was extracted for Droplet Digital PCR analysis. Among the investigated siRNAs we identified four effective candidates against all three segments of CCHF genome: one for the S and M segments, whilst two for the L segment. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs in vitro. The most active siRNAs were demonstrated a specific inhibitory effect against CCHFV in a dose-dependent manner. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be used in future anti-CCHFV therapy developments.

scholarly journals Cold priming uncouples light- and cold-regulation of gene expression in Arabidopsis thaliana

2020 ◽  
Author(s):  
Andras Bittner ◽  
Jörn van Buer ◽  
Margarete Baier
Keyword(s):  
Gene Expression ◽  
Arabidopsis Thaliana ◽  
Plant Pathogens ◽  
Cold Treatment ◽  
Regulation Of Gene Expression ◽  
Light Regulation ◽  
High Light ◽  
Cold Stimulus ◽  
Expression Of Genes ◽  
Specific Manner

Abstract Background: The majority of stress-sensitive genes responds to cold and high light in the same direction, if plants face the stresses for the first time. As shown recently for a small selection of genes of the core environmental stress response cluster, pre-treatment of Arabidopsis thaliana with a 24 h long 4 °C cold stimulus modifies cold regulation of gene expression for up to a week at 20 °C, although the primary cold effects are reverted within the first 24 h. Such memory-based regulation is called priming. Here, we analyse the effect of 24 h cold priming on cold regulation of gene expression on a transcriptome-wide scale and investigate if and how cold priming affects light regulation of gene expression.Results: Cold-priming affected cold and excess light regulation of a small subset of genes. In contrast to the strong gene co-regulation observed upon cold and light stress in not-primed plants, most priming-sensitive genes were regulated in a stressor-specific manner in cold-primed plant. Furthermore, almost as much genes were inversely regulated as co-regulated by a 24 h long 4 °C cold treatment and exposure to heat-filtered high light (800 µmol quanta m-2 s-1). Gene ontology enrichment analysis revealed that cold priming preferentially supports expression of genes involved in the defence against plant pathogens upon cold triggering. The regulation took place on the cost of the expression of genes involved in growth regulation and transport. On the contrary, cold priming resulted in stronger expression of genes regulating metabolism and development and weaker expression of defence genes in response to high light triggering. qPCR with independently cultivated and treated replicates confirmed the trends observed in the RNASeq guide experiment.Conclusion: A 24 h long priming cold stimulus activates a several days lasting stress memory that controls cold and light regulation of gene expression and adjusts growth and defence regulation in a stressor-specific manner.

Suppression of microRNA accumulation via RNA interference in Arabidopsis thaliana

2010 ◽  
Vol 73 (4-5) ◽  
pp. 391-397 ◽  
Author(s):  
Fabián E. Vaistij ◽  
Luisa Elias ◽  
Gilu L. George ◽  
Louise Jones
Keyword(s):  
Arabidopsis Thaliana ◽  
Rna Interference

RNA Interference Mechanisms and Applications in Plant Pathology

2018 ◽  
Vol 56 (1) ◽  
pp. 581-610 ◽  
Author(s):  
Cristina Rosa ◽  
Yen-Wen Kuo ◽  
Hada Wuriyanghan ◽  
Bryce W. Falk
Keyword(s):  
Plant Pathology ◽  
Rna Interference ◽  
Gene Function ◽  
Small Rnas ◽  
Plant Pathogens ◽  
Common Ancestor ◽  
Plant Protection ◽  
Plant Diseases ◽  
Antiviral Defense ◽  
Endogenous Genes

The origin of RNA interference (RNAi), the cell sentinel system widely shared among eukaryotes that recognizes RNAs and specifically degrades or prevents their translation in cells, is suggested to predate the last eukaryote common ancestor ( 138 ). Of particular relevance to plant pathology is that in plants, but also in some fungi, insects, and lower eukaryotes, RNAi is a primary and effective antiviral defense, and recent studies have revealed that small RNAs (sRNAs) involved in RNAi play important roles in other plant diseases, including those caused by cellular plant pathogens. Because of this, and because RNAi can be manipulated to interfere with the expression of endogenous genes in an intra- or interspecific manner, RNAi has been used as a tool in studies of gene function but also for plant protection. Here, we review the discovery of RNAi, canonical mechanisms, experimental and translational applications, and new RNA-based technologies of importance to plant pathology.

scholarly journals Mechanism and Function of Antiviral RNA Interference in Mice

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Qingxia Han ◽  
Gang Chen ◽  
Jinyan Wang ◽  
David Jee ◽  
Wan-Xiang Li ◽  
...  
Keyword(s):  
Rna Interference ◽  
Rna Replication ◽  
Interferon Response ◽  
Type I ◽  
Small Interfering Rnas ◽  
Guide Rna ◽  
Argonaute 2 ◽  
Adult Mice ◽  
And Function ◽  
Nodamura Virus

ABSTRACT Distinct mammalian RNA viruses trigger Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNA) and encode unrelated proteins to suppress vsiRNA biogenesis. However, the mechanism and function of the mammalian RNA interference (RNAi) response are poorly understood. Here, we characterized antiviral RNAi in a mouse model of infection with Nodamura virus (NoV), a mosquito-transmissible positive-strand RNA virus encoding a known double-stranded RNA (dsRNA)-binding viral suppressor of RNAi (VSR), the B2 protein. We show that inhibition of NoV RNA replication by antiviral RNAi in mouse embryonic fibroblasts (MEFs) requires Dicer-dependent vsiRNA biogenesis and Argonaute-2 slicer activity. We found that VSR-B2 of NoV enhances viral RNA replication in wild-type but not RNAi-defective MEFs such as Argonaute-2 catalytic-dead MEFs and Dicer or Argonaute-2 knockout MEFs, indicating that VSR-B2 acts mainly by suppressing antiviral RNAi in the differentiated murine cells. Consistently, VSR-B2 expression in MEFs has no detectable effect on the induction of interferon-stimulated genes or the activation of global RNA cleavages by RNase L. Moreover, we demonstrate that NoV infection of adult mice induces production of abundant vsiRNA active to guide RNA slicing by Argonaute-2. Notably, VSR-B2 suppresses the biogenesis of both vsiRNA and the slicing-competent vsiRNA-Argonaute-2 complex without detectable inhibition of Argonaute-2 slicing guided by endogenous microRNA, which dramatically enhances viral load and promotes lethal NoV infection in adult mice either intact or defective in the signaling by type I, II, and III interferons. Together, our findings suggest that the mouse RNAi response confers essential protective antiviral immunity in both the presence and absence of the interferon response. IMPORTANCE Innate immune sensing of viral nucleic acids in mammals triggers potent antiviral responses regulated by interferons known to antagonize the induction of RNA interference (RNAi) by synthetic long double-stranded RNA (dsRNA). Here, we show that Nodamura virus (NoV) infection in adult mice activates processing of the viral dsRNA replicative intermediates into small interfering RNAs (siRNAs) active to guide RNA slicing by Argonaute-2. Genetic studies demonstrate that NoV RNA replication in mouse embryonic fibroblasts is inhibited by the RNAi pathway and enhanced by the B2 viral RNAi suppressor only in RNAi-competent cells. When B2 is rendered nonexpressing or nonfunctional, the resulting mutant viruses become nonpathogenic and are cleared in adult mice either intact or defective in the signaling by type I, II, and III interferons. Our findings suggest that mouse antiviral RNAi is active and necessary for the in vivo defense against viral infection in both the presence and absence of the interferon response.

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